Humanized il-15 animals

ABSTRACT

Genetically modified non-human animals comprising a humanized interleukin-15 (IL-15) gene. Cells, embryos, and non-human animals comprising a human IL-15 gene. Rodents that express humanized or human IL-15 protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/732,400, filed Jan. 2, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/832,976, filed Dec. 6, 2017, now U.S. Pat. No.10,561,125, which is a continuation of U.S. patent application Ser. No.14/842,342, filed Sep. 1, 2015, now abandoned, which is a continuationof U.S. patent application Ser. No. 14/514,454, filed Oct. 15, 2014, nowU.S. Pat. No. 9,155,290, which claims the benefit of priority of U.S.Provisional Application No. 61/891,013, filed Oct. 15, 2013, the entirecontents of which are incorporated herein by reference.

FIELD

Non-human animals comprising in their germline a humanized endogenousnon-human IL-15 locus. Non-human animals comprising in their germline ahumanized IL-15-encoding sequence under control of endogenous non-humanregulatory elements. Non-human animals (e.g., mammals, e.g., rodentssuch as mice, rats, and hamsters) that comprise a genetic modificationcomprising a replacement, at an endogenous locus, of a non-human IL-15gene sequence with a human or humanized IL-15 gene sequence. Rodents andother non-human animals that express human or humanized IL-15 from anendogenous modified non-human IL-15 locus. Non-human animals thatexpress human or humanized IL-15 under the control of a non-human IL-15promoter and/or regulatory sequences.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The sequence listing in the XML, named as31013ZYXW_2200US05_SequenceListing.xml of 14 KB, created on Sep. 30,2022, and submitted to the United States Patent and Trademark Office viaPatent Center, is incorporated herein by reference.

BACKGROUND

Transgenic mice with randomly inserted transgenes that contain a humanIL-15 sequence are known in the art. However, transgenic mice thatexpress human IL-15 from randomly integrated transgenes are not optimalin one respect or another. For example, most mice transgenic for humanIL-15 exhibit abnormal levels and/or ratios of certain cells, includinglymphocytes (e.g., T cells), that are likely due to a dysregulation ofimmune cell function. Such mice also exhibit a panoply of pathologies,presumably ultimately due to dysregulation of the transgenic IL-15. Suchdysregulation may result from, e.g., absence of endogenous controlelements, and/or placement of the human IL-15 sequence away from theendogenous IL-15 locus.

There remains a need in the art for non-human animals that comprisehuman IL-15-encoding sequences, wherein the human IL-15 encodingsequences are at an endogenous non-human IL-15 locus, and/or are underregulatory control of endogenous non-human IL-15 elements (e.g.,upstream and/or downstream noncoding regions). There is a need in theart for non-human animals that express human IL-15 under the control ofendogenous non-human regulatory elements. There is a need in the art fornon-human animals that express human IL-15 in a manner that isphysiologically relevant in the non-human animal. There is a need in theart for non-human animals that express a human IL-15, wherein thenon-human animals lack a significant abnormality in lymphocytepopulations, e.g., in T cell populations. There is also a need in theart for non-human animals that express human or humanized IL-15, andthat lack one or more of the pathologies exhibited by non-human animalsthat are transgenic for human IL-15.

SUMMARY

In various aspects and embodiments, genetically modified non-humanorganisms comprising a humanized IL-15 locus are provided. Non-humanorganisms that comprise a humanized IL-15 gene are provided, wherein thehumanized IL-15 gene is under control of one or more endogenousnon-human regulatory elements. Non-human organisms that comprise ahumanized IL-15 gene at an endogenous non-human IL-15 locus areprovided. Non-human organisms that comprise an endogenous humanizedIL-15 locus that is capable of being passed through the germline of theorganisms are provided. Non-human animals, e.g., mammals (e.g., rodents,e.g., mice or rats) that express human IL-15 from a modified endogenousIL-15 locus are provided, wherein the expressed IL-15 is fully or partlyhuman.

Genetically modified non-human animals, embryos, cells, tissues, andnucleic acids are provided, which comprise a human IL-15 genomicsequence regulated by non-human IL-15 regulatory control. The non-humananimals express a humanized IL-15 protein, or fully human IL-15 protein(e.g., a fully human mature IL-15 protein), and do not exhibit one ormore of the pathologies of transgenic human IL-15 non-human animalsknown in the art. In various embodiments, the non-human animals aremammals, e.g., rodents, e.g., mice, rats, hamsters, etc. In a specificembodiment, the mammal is a rodent; in another specific embodiment, therodent is a mouse or a rat.

Genetically modified non-human animals, embryos, cells, tissues, andnucleic acids are provided, which comprise a human IL-15 genomicsequence at an endogenous non-human IL-15 locus. The non-human animalsexpress a humanized IL-15 protein, or fully human IL-15 protein (e.g., afully human mature IL-15 protein), from a modified endogenous non-humanlocus regulated by one or more endogenous non-human regulatory sequencesof the modified endogenous IL-15 locus, and do not exhibit one or moreof the pathologies of transgenic human IL-15 non-human animals known inthe art. In various embodiments, the non-human animals are mammals,e.g., rodents, e.g., mice, rats, hamsters, etc. In a specificembodiment, the mammal is a rodent; in another specific embodiment, therodent is a mouse or a rat.

In various embodiments and aspects, the non-human animals comprise amodified IL-15 locus in the genome of the non-human animal such that themodified IL-15 locus is capable of being passed through the germline,wherein the modified endogenous IL-15 locus comprises a humanization ofat least a mature protein-coding portion of the endogenous IL-15 locus.In various embodiments, the non-human animals are mammals. In variousembodiments, the mammals are rodents, and the rodents are heterozygousor homozygous with respect to the modified IL-15 locus. In variousembodiments, the rodents are selected from mice and rats. In variousembodiments, the mice and rats are homozygous for the modified IL-15locus, and are incapable of expressing an endogenous fully mouse orfully rat IL-15 protein, and the mice and rats express a mature humanIL-15 protein.

In one embodiment, a non-human animal is provided that comprises a firstendogenous wild-type IL-15 allele, and a humanization of a secondendogenous IL-15 allele.

In one embodiment, a non-human animal is provided that comprises a lackof a first endogenous IL-15 allele and a humanization of a secondendogenous IL-15 allele.

In one embodiment, a non-human animal is provided that comprises a lackof a functional endogenous IL-15 allele, and comprise at least one copyof a humanized IL-15 allele under the control of endogenous non-humanregulatory elements. In one embodiment, the at least one copy of ahumanized IL-15 allele is at an endogenous IL-15 locus.

In various embodiments and aspects, the humanization is of one or moreexons and/or introns at the endogenous non-human IL-15 locus. In variousembodiments and aspects, non-human animals having a modified IL-15 locusare provided wherein one or both of an endogenous non-human5′-untranslated region and an endogenous non-human 3′-untranslatedregion are retained in the modified non-human animal.

In one embodiment, the humanization of the endogenous non-human IL-15locus is with a coding region (or fragment thereof) that is a genomicfragment of a human IL-15 locus that comprises at least one human IL-15protein-coding exon.

In one embodiment, the humanization of the endogenous non-human IL-15locus is with a coding region that is a genomic fragment of a humanIL-15 locus that comprises each human IL-15 protein-coding exon, butdoes not comprise a non-human IL-15 protein-coding exon.

In one embodiment, the IL-15 locus that comprises the human genomicfragment results in the expression of an IL-15 protein that when matureis fully human. In one embodiment, the humanization of the endogenousnon-human IL-15 locus is with a cDNA encoding a human IL-15 protein,such that upon processing in the non-human animal the mature IL-15protein produced by the humanized locus is fully human.

In one aspect, a genetically modified non-human animal is provided thatcomprises an endogenous IL-15 locus that is humanized in whole or inpart, wherein the humanized IL-15 locus comprises a humanizedIL-15-coding gene that is under control of endogenous non-humanregulatory elements. In one embodiment, the endogenous non-humanregulatory elements comprise all endogenous IL-15 regulatory elementsupstream (with respect to transcriptional direction of the IL-15 gene)of the first protein-coding region or exon of the humanized IL-15 gene.In one embodiment, the endogenous non-human regulatory elements compriseall endogenous IL-15 regulatory elements downstream (with respect totranscriptional direction of the IL-15 gene) of the last protein-codingregion or exon on the humanized IL-15 gene. In one embodiment, thehumanized IL-15-coding gene comprises a human 3′UTR.

In one aspect, a genetically modified rodent is provided that comprisesa replacement at an endogenous rodent IL-15 locus of an endogenousrodent IL-15 genomic sequence with a human IL-15 genomic sequence. Inone embodiment, the genetic modification is in the germline of thenon-human animal.

In one embodiment, the genetically modified rodent comprises a firstrodent regulatory sequence upstream (with respect to the direction oftranscription of the IL-15 gene) of the human IL-15 genomic sequence anda second rodent regulatory sequence downstream of the human IL-15genomic sequence. In one embodiment, the first rodent regulatorysequence comprises a rodent promoter and/or enhancer, and the secondrodent regulatory sequence comprises a 3′-UTR.

In one aspect, a genetically modified non-human animal is provided thatexpresses a human or humanized IL-15 protein

In one aspect, a genetically modified mouse is provided that comprises areplacement at an endogenous mouse IL-15 locus of an endogenous mouseIL-15 genomic sequence (or fragment thereof) with a human IL-15 genomicsequence (or fragment thereof) to form a modified locus, wherein thehuman IL-15 genomic sequence comprises at least one human protein-codingexon.

In one embodiment, the replacement comprises a human genomic fragmentcomprising at least two protein-coding exons of human IL-15. In oneembodiment, the replacement comprises a human genomic fragment thatcomprises at least three protein-coding exons of human IL-15. In oneembodiment, the replacement comprises a human genomic fragment thatcomprises at least four protein-coding exons of human IL-15. In oneembodiment, the replacement comprises a human genomic fragment thatcomprises protein-coding exons 3, 4, 5 and 6 of human IL-15. In oneembodiment, the replacement comprises less than all human IL-15 exons,wherein the human exons of the replacement consist of thedownstream-most (with respect to direction of transcription of the IL-15gene) four protein-coding exons of the human IL-15 gene. In oneembodiment, the replacement consists essentially of a human genomicfragment that contains no more than four protein-coding exons of humanIL-15; in one embodiment, the replacement further consists essentiallyof human intronic sequence upstream of the 5′-most human exon and humannon-protein-coding sequence downstream of the human stop codon anddownstream of the human 3′UTR.

In one aspect, a genetically modified mouse is provided that comprises ahumanized IL-15 locus, wherein the humanized IL-15 locus comprisesnon-protein-coding mouse exons, wherein each (mature) protein-codingmouse exon is replaced with (mature) protein-coding human exons. In oneembodiment, the humanized IL-15 locus comprises a replacement of a mousegenomic fragment that encodes mature (i.e., non pre-protein) mouse IL-15protein sequences with a human genomic fragment that encodes mature(i.e., non-preprotein) human IL-15 protein sequences.

In one aspect, a genetically modified mouse is provided that comprises asequence that is at least 95%, 96%, 97%, 98%, or 99% identical, or isidentical, to SEQ ID NO:5.

In one aspect, a genetically modified mouse is provided that comprises asequence that is at least 95%, 96%, 97%, 98%, or 99% identical, or isidentical, to SEQ ID NO:5; wherein the mouse lacks an endogenoussequence encoding exons 3 through 6 of a mouse IL-15 protein as depictedherein, and the mouse comprises a nucleic acid sequence at an endogenousmouse IL-15 locus wherein the nucleic acid sequence encodes human IL-15exons 3, 4, 5, and 6 as depicted herein.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein from an endogenous mouse IL-15 locusthat is modified to comprise at least one human IL-15 exon that encodesamino acids in a mature human IL-15 protein. In one embodiment, theendogenous rodent IL-15 locus comprises at least two human IL-15 exonsthat encode amino acids in a mature human IL-15 protein. In oneembodiment, the endogenous rodent IL-15 locus comprises at least threehuman IL-15 exons that encode amino acids in a mature human IL-15protein. In one embodiment, the endogenous rodent IL-15 locus comprisesat least four human IL-15 exons that encode amino acids in a maturehuman IL-15 protein. In one embodiment, the endogenous rodent compriseshuman IL-15 exons 3, 4, 5 and 6 that encode amino acids in a maturehuman IL-15 protein. In one embodiment, the endogenous rodent IL-15locus comprises all human nucleic acid sequence that encodes amino acidsin a mature human IL-15 protein.

In one embodiment, the humanization comprises a human IL-15 3′UTR. Inone embodiment, the rodent locus comprises at least one exon that doesnot encode amino acids of a mature IL-15 protein or at least one exonthat includes a nucleotide sequence that does not encode amino acids ofa mature IL-15 protein. In one embodiment, the rodent locus comprises atleast two exons that do not encode amino acids of a mature IL-15protein. In one embodiment, the rodent locus comprises at least threeexons that do not encode amino acids of a mature IL-15 protein. In oneembodiment, the rodent locus comprises four exons that do not encodeamino acids of a mature IL-15 protein.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the lymphocyte population of therodent is characterized by its T cell population that is about the samein number as a population of T cells in an age-matched wild-type mouse.In one embodiment, the modified rodent is characterized by a populationof mature T cells that is about the same in number as a population ofmature T cells in an age-matched wild-type mouse.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the lymphocyte population of therodent is characterized by a population of T cells that is about thesame in number as a population of T cells in an age-matched wild-typemouse. In one embodiment, the modified rodent exhibits a population ofmature T cells that is about the same in number as a population ofmature T cells in an age-matched wild-type mouse. In one embodiment, themodified rodent exhibits a population of peripheral T cells that isabout the same in number as the population of peripheral T cells in anage-matched wild-type mouse. In one embodiment, the mature humanizedIL-15 protein is identical to a mature human IL-15 protein.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the lymphocyte population of therodent is characterized by a T cell population that exhibits a CD4:CD8ratio that is about the same as the CD4:CD8 ratio in the T cellpopulation of an age-matched wild-type mouse. In one embodiment, thehumanized IL-15 protein is identical to a human IL-15 protein.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the lymphocyte population of therodent is characterized by a natural killer (NK) cell population that isabout the same in size as an NK cell population of an age-matchedwild-type mouse. In one embodiment, the humanized IL-15 protein isidentical to a human IL-15 protein.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the lymphocyte population ofthe rodent is characterized by a T cell population and an NK cellpopulation that are each about the same in size as a T cell populationand an NK cell population in an age-matched wild-type mouse.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not developspontaneous intestinal inflammation. In one embodiment, the rodent doesnot display a propensity to develop intestinal inflammation any morethan an age-matched wild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not developspontaneous inflammation in the duodeno-jejunal area. In one embodiment,the rodent does not display a propensity to develop inflammation in theduodeno-jejunal area any more than an age-matched wild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not exhibitdestruction of intestinal epithelium greater than an age-matchedwild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not exhibit celiacdisease at a higher rate or frequency than an age-matched wild-typerodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not exhibit a higherresistance to diet-induced adiposity, and does not exhibit a higherinsulin sensitivity, than an age-matched wild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent upon infection with apathogen is no more susceptible to lipopolysaccharide-induced lethalliver injury than an age-matched wild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not develop psoriaticlesions at a higher rate or frequency than an age-matched wild-typerodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not develop arthritisat a higher rate or frequency than an age-matched wild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not developlymphocyte infiltration of joints at a higher rate or frequency than anage-matched wild-type rodent.

In one aspect, a genetically modified rodent is provided that expressesa humanized IL-15 protein, wherein the rodent does not developinflammatory synovitis at a rate higher than an age-matched wild-typecontrol rodent.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the rodent does not developone or more pathologies at a rate or frequency higher than that of anage-matched wild-type rodent, wherein the one or more pathologies isselected from the group consisting of arthritis, lymphocyte infiltrationof joints, inflammatory synovitis, psoriatic lesions, pathogen-relatedlipopolysaccharide-induced lethal liver injury, a resistance to insulin,a resistance to diet-induced adiposity, spontaneous intestinalinflammation, spontaneous inflammation in the duodeno-jejunal area,destruction of intestinal epithelium, celiac disease, and a combinationthereof.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the rodent does not developtwo or more pathologies at a rate or frequency higher than that of anage-matched wild-type rodent, wherein the two or more pathologies isselected from the group consisting of arthritis, lymphocyte infiltrationof joints, inflammatory synovitis, psoriatic lesions, pathogen-relatedlipopolysaccharide-induced lethal liver injury, a resistance to insulin,a resistance to diet-induced adiposity, spontaneous intestinalinflammation, spontaneous inflammation in the duodeno-jejunal area,destruction of intestinal epithelium, and celiac disease.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the rodent does not developthree or more pathologies at a rate or frequency higher than that of anage-matched wild-type rodent, wherein the three or more pathologies isselected from the group consisting of arthritis, lymphocyte infiltrationof joints, inflammatory synovitis, psoriatic lesions, pathogen-relatedlipopolysaccharide-induced lethal liver injury, a resistance to insulin,a resistance to diet-induced adiposity, spontaneous intestinalinflammation, spontaneous inflammation in the duodeno-jejunal area,destruction of intestinal epithelium, and celiac disease.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the rodent does not developfour or more pathologies at a rate or frequency higher than that of anage-matched wild-type rodent, wherein the four or more pathologies isselected from the group consisting of arthritis, lymphocyte infiltrationof joints, inflammatory synovitis, psoriatic lesions, pathogen-relatedlipopolysaccharide-induced lethal liver injury, a resistance to insulin,a resistance to diet-induced adiposity, spontaneous intestinalinflammation, spontaneous inflammation in the duodeno-jejunal area,destruction of intestinal epithelium, and celiac disease.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the rodent does not developfive or more pathologies at a rate or frequency higher than that of anage-matched wild-type rodent, wherein the five or more pathologies isselected from the group consisting of arthritis, lymphocyte infiltrationof joints, inflammatory synovitis, psoriatic lesions, pathogen-relatedlipopolysaccharide-induced lethal liver injury, a resistance to insulin,a resistance to diet-induced adiposity, spontaneous intestinalinflammation, spontaneous inflammation in the duodeno-jejunal area,destruction of intestinal epithelium, and celiac disease.

In one aspect, a genetically modified rodent is provided that expressesa human or humanized IL-15 protein, wherein the rodent does not developsix or more pathologies at a rate or frequency higher than that of anage-matched wild-type rodent, wherein the six or more pathologies isselected from the group consisting of arthritis, lymphocyte infiltrationof joints, inflammatory synovitis, psoriatic lesions, pathogen-relatedlipopolysaccharide-induced lethal liver injury, a resistance to insulin,a resistance to diet-induced adiposity, spontaneous intestinalinflammation, spontaneous inflammation in the duodeno-jejunal area,destruction of intestinal epithelium, and celiac disease.

In one aspect, a genetically modified non-human animal is provided thatcomprises a humanization of an endogenous IL-15 locus, wherein theanimal expresses a partly or fully-human mature IL-15 protein, andwherein the partly of fully-human mature IL-15 protein is expressed atcomparable levels and in the same tissues as an endogenous IL-15 proteinin an age-matched wild-type animal.

In one aspect, a large targeting vector (LTVEC) is provided thatcomprises homology arms to an endogenous non-human IL-15 locus, whereinthe LTVEC comprises, disposed between said homology arms, a contiguoushuman genomic fragment comprising protein-coding exons of a human IL-15gene. In one embodiment, the contiguous human genomic fragment does notcomprise non-protein-coding exons of a human IL-15 locus. In oneembodiment, the contiguous human genomic fragment further comprises ahuman 3′UTR of an IL-15 gene.

In one aspect, a nucleic acid (e.g., DNA) construct is provided,comprising from 5′ to 3′ with respect to direction of transcription, anucleic acid sequence homologous to a mouse IL-15 5′ noncoding sequence,a human genomic fragment comprising human IL-15 protein-encoding exonsbut not comprising a human regulatory sequence upstream with respect tothe human IL-15 protein-encoding sequence, and a nucleic acid sequencehomologous to a mouse IL-15 3′ noncoding sequence. In one embodiment,the human genomic fragment further comprises a human IL-15 3′UTR.

In one aspect, a nucleic acid (e.g., DNA) construct is provided,comprising from 5′ to 3′ with respect to direction of transcription, anucleic acid sequence that comprises a region of homology to mouse IL-15gene sequences upstream of the first mouse IL-15 protein-coding exon, ahuman genomic fragment encoding a human IL-15 protein but not comprisinga human regulatory sequence upstream of sequence encoding the humanIL-15 protein, and a nucleic acid sequence homologous to a mouse IL-153′ noncoding sequence. In one embodiment, the human genomic fragmentfurther comprises a human IL-15 3′UTR.

In one aspect, a genetically modified non-human cell is provided,wherein the non-human cell comprises a replacement at an endogenousnon-human IL-15 locus of a gene sequence encoding a non-human IL-15 witha human genomic sequence encoding a human IL-15.

In one embodiment, the human genomic sequence comprises a contiguoushuman nucleic acid sequence spanning all protein-coding exons of thehuman IL-15 gene.

In one embodiment, the genetically modified rodent comprises a non-humanIL-15 promoter at the endogenous non-human IL-15 locus.

In one embodiment, the genetically modified non-human animal comprisesall rodent non-protein-coding exons and regulatory regions upstream ofthe first protein-coding exon of the rodent IL-15 gene.

In one embodiment, the cell is selected from a pluripotent cell, aninduced pluripotent cell, a totipotent cell, an ES cell, a somatic cell,and an ovum.

In one embodiment, the cell expresses human IL-15 protein.

In one embodiment, the non-human animal is a mammal. In one embodiment,the mammal is a rodent. In one embodiment, the rodent is selected from amouse and a rat.

In one aspect, a non-human embryo is provided, wherein the embryocomprises at least one non-human donor cell (e.g., an ES cell, apluripotent cell, a totipotent cell, etc.) comprising a replacement ofan endogenous non-human IL-15-encoding nucleic acid sequence with ahuman IL-15-encoding nucleic acid sequence at an endogenous non-humanIL-15 locus. In one embodiment, the donor cell is a non-human ES celland the embryo is a host non-human animal embryo that is a pre-morula, amorula, or a blastocyst.

In one embodiment, the non-human embryo is a rat embryo, and the atleast one non-human donor cell is a rat cell. In one embodiment, thenon-human ES cell is a rat ES cell and the host embryo is a rat embryo.

In one embodiment, the non-human embryo is a mouse embryo, and the atleast one non-human donor cell is a mouse cell. In one embodiment, thenon-human ES cell is a mouse ES cell and the host embryo is a mouseembryo.

In one aspect, a rodent tissue that comprises a humanized IL-15 gene atan endogenous rodent IL-15 locus is provided. In one embodiment, thetissue is selected from epithelial tissue, skin tissue, and muscletissue.

In one aspect, a genetically modified rodent is provided that comprisesa nucleic acid (e.g., DNA) sequence that encodes a human IL-15 protein,wherein the rodent does not express a rodent IL-15, and wherein therodent exhibits an NK cell population that is about the same size as anNK cell population of a wild-type rodent. In one embodiment, the rodentis a rat. In one embodiment, the rodent is a mouse.

In one embodiment, the rodent exhibits a peripheral T cell populationthat is about the same size as a peripheral T cell population of anage-matched wild-type rodent.

In one aspect, a method is provided for making a non-human animal thatexpresses a human or humanized IL-15 protein, comprising geneticallymodifying a non-human animal as described herein to form a nucleic acidsequence in the non-human animal that comprises a nucleic acid sequence(e.g., DNA) that encodes a human or humanized IL-15 protein, wherein thenucleic acid sequence is under control of endogenous non-human upstreamand downstream regulatory elements.

In some embodiments, the non-human animal is genetically modified byreplacing an endogenous IL-15 genomic sequence (or fragment thereof), atan endogenous IL-15 locus, with a human IL-15 genomic sequence (orfragment thereof) to form a modified locus, wherein the human IL-15genomic sequence comprises at least one human protein-coding exon. Inone embodiment, the replacement comprises a human genomic fragmentcomprising at least two protein-coding exons of human IL-15. In oneembodiment, the replacement comprises a human genomic fragment thatcomprises at least three protein-coding exons of human IL-15. In oneembodiment, the replacement comprises a human genomic fragment thatcomprises at least four protein-coding exons of human IL-15. In aspecific embodiment, the replacement comprises a human genomic fragmentthat comprises protein-coding exons 3, 4, 5 and 6 of human IL-15. In oneembodiment, the replacement further comprises human non-protein-codingsequence downstream of the human stop codon (e.g., the human 3′UTR).

In one embodiment, the non-human animal is produced from a pluripotentor totipotent cell (e.g., an ES cell). In one embodiment, the non-humananimal is produced employing a nuclear injection step wherein a nucleicacid construct comprising the humanized IL-15 gene (optionally withupstream and/or downstream endogenous non-human regulatory sequences) isintroduced by pronuclear injection. In one embodiment, the nucleic acidconstruct comprises a human genomic fragment that comprisesprotein-coding exons 3, 4, 5 and 6 of human IL-15. In one embodiment,the nucleic acid construct further comprises human non-protein-codingsequence downstream of the human stop codon (e.g., the human 3′UTR). Inone embodiment, the non-human animal is produced employing a non-humanfibroblast that is genetically modified with a human or humanized IL-15gene and (optionally) upstream and/or downstream non-human IL-15regulatory elements.

In one aspect, a method for identifying an agent that is an antagonistof human IL-15 is provided, comprising a step of administering an agentto a genetically modified rodent as described herein, determining aneffect of the agent on a human IL-15-mediated function in the rodent,and identifying the agent as an IL-15 antagonist if it antagonizes thefunction of human IL-15 in the genetically modified rodent.

In one embodiment, the agent comprises an immunoglobulin variable domainthat binds IL-15. In one embodiment, the agent specifically binds humanIL-15 but not rodent IL-15. In one embodiment, the agent is an antibody.

In one aspect, a method for determining whether an agent reducesIL-15-mediated lymphocyte development is provided, comprising a step ofadministering to a genetically modified rodent as described herein anIL-15 antagonist for a period of time, measuring a lymphocyte number ofthe rodent at one or more time points following administration, anddetermining whether the IL-15 antagonist reduces the lymphocytepopulation.

In one aspect a method for determining whether an agent reducesIL-15-mediated lymphocyte infiltration of a tissue or a joint isprovided, comprising a step of administering to a genetically modifiedrodent as described herein an IL-15 antagonist for a period of time,measuring lymphocyte infiltration of the tissue or the joint at one ormore time points following administration, an determining whether theIL-15 antagonist reduces lymphocyte infiltration of the tissue or joint.

In one aspect, a method is provided for determining whether an agentreduces IL-15-mediated arthritic progression, comprising a step ofadministering to a genetically modified rodent as described herein, andfurther comprising an induced arthritis, an IL-15 antagonist for aperiod of time, measuring arthritic progression, and determining whetherthe IL-15 antagonist affects arthritic progression in the rodent.

Unless otherwise stated, or apparent from the context, two or moreaspects and/or embodiments can be combined.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts (not to scale) a schematic of a wild-type mouse IL-15gene locus (top) and a humanized endogenous mouse IL-15 locus (bottom).Open symbols indicate human sequence; closed symbols indicate mousesequence; stippled items indicate untranslated regions. Upstream (to theleft in the figure) noncoding exons of the mouse IL-15 gene are notshown (and were not humanized). The bottom construct depicts anembodiment of a humanized IL-15 gene comprising a humanized 3′UTR(stippled) and a removable drug selection cassette, which is optionallyremoved in the humanized animal.

FIG. 2 is a nucleic acid sequence (SEQ ID NO:1) that depicts theupstream (with respect to direction of transcription of the IL-15 gene)junction between mouse sequence and human sequence; the sequence shownbegins with mouse sequence in uppercase, followed by an AsisIrestriction site in lowercase, followed by human IL-15 nucleic acidsequence in uppercase. The ellipses indicate that sequence continuesupstream and downstream of the sequence shown.

FIG. 3 is an embodiment of a nucleic acid sequence (SEQ ID NO:2)indicating downstream human IL-15 coding and noncoding sequence inuppercase (human 3′UTR underscored), followed by an XhoI site inlowercase, followed by a lox site (uppercase, underscored), followed bysequence of the downstream neo selection cassette (uppercase), whichextends 2.6 kb downstream.

FIG. 4 is a nucleic acid sequence (SEQ ID NO:3) that depicts thejunction between the downstream portion of the neo selection cassette(uppercase), with lox site (uppercase and underscored), followed by anNheI site (lowercase), which is followed by mouse sequence downstream ofthe humanization (uppercase); the 2.6 kb indicates that the selectioncassette extends further upstream; ellipses indicate that sequencecontinues.

FIG. 5 depicts an alignment of the protein sequences for the mouse IL-15precursor protein (top, “mIL15_precursor,” SEQ ID NO:4); the hybridmouse/human IL-15 precursor protein (middle, “Regn_Hybrid_m/h,” SEQ IDNO:5) produced by a humanization of the locus depicted in FIG. 1 ; andthe known human IL-15 isoform 1 preproprotein (bottom, “hIL15 isoform1,” SEQ ID NO:6); the junction resulting from the humanization asdepicted in FIG. 1 is indicated by a vertical arrow indicating C as thefirst amino acid of the replacement; the mature hIL-15 protein start isindicated by the bent arrow at the first amino acid N.

FIG. 6 depicts IL-15 detected in serum from poly I:C injectedheterozygous hIL-15 mice. The control indicates that no hIL-15 wasdetected in cultured splenocytes from poly I:C injected wild-type mice.

FIG. 7 depicts that human IL-15 does not react with mouse IL-15 or polyI:C in an ELISA assay. Mouse IL-15 was at 1000 μg/mL.

FIG. 8 depicts BM-DCs from mice heterozygous for human IL-15, using7-fold concentrated BM-DC supernatants for untreated mice, poly I:Ctreated mice, and LPS treated mice.

FIG. 9 depicts that BM-DCs from mice heterozygous for human IL-15produce human IL-15 transcript (RT-PCR data). Lane 1: untreated mice;Lane 2: poly I:C treated mice; Lane 3: LPS only; Lane 4: wild-typeuntreated mice; Lane 5: wild-type poly I:C treated mice; Lane 6:wild-type LPS treated mice; Lane 7: no cDNA control (water only).

FIG. 10 depicts that MAID 5218 het mice produced human IL-15 upon polyI:C injection at a level comparable to MAID 5217 het mice.

DETAILED DESCRIPTION

Genetically modified non-human organisms are provided that comprise amodified endogenous IL-15 locus that comprises a human sequence, e.g., areplacement of one or more non-human sequences with one or more humansequences. The organisms are generally able to pass the modification toprogeny, i.e., through germline transmission. In particular, geneticallymodified non-human animals are provided.

The genetically modified non-human animal may be selected from a groupconsisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull,buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g.,marmoset, rhesus monkey). For the non-human animals where suitablegenetically modifiable ES cells are not readily available, other methodsare employed to make a non-human animal comprising the geneticmodification. Such methods include, e.g., modifying a non-ES cell genome(e.g., a fibroblast or an induced pluripotent cell) and employingnuclear transfer to transfer the modified genome to a suitable cell,e.g., an oocyte, and gestating the modified cell (e.g., the modifiedoocyte) in a non-human animal under suitable conditions to form anembryo.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm),129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al.(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.In yet another embodiment, the mouse is of a hybrid line (e.g., 50%BALB/c—50% 12954/Sv; or 50% C57BL/6—50% 129; e.g., F1H4 cells, see,e.g., Auerbach et al. (2000)).

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

The non-human animal may have one or more other genetic modifications,and/or other modifications, that are suitable for the particular purposefor which the humanized IL-15 mouse is made. For example, suitable micefor maintaining a xenograft (e.g., a human cancer or tumor), maycomprise one or more modifications that compromise, inactivate, ordestroy the immune system of the non-human animal in whole or in part.Compromise, inactivation, or destruction of the immune system of thenon-human animal can include, for example, destruction of hematopoieticcells and/or immune cells by chemical means (e.g., administering atoxin), physical means (e.g., irradiating the animal), and/or geneticmodification (e.g., knocking out one or more genes). Non-limitingexamples of such mice may include, e.g., NOD mice, SCID mice, NON/SCIDmice, IL2Rγ knockout mice, NOD/SCID/γc^(null) mice (see, e.g., Ito, M.et al. (2002) NOD/SCID/γcnull mouse: an excellent recipient mouse modelfor engraftment of human cells, Blood 100(9):3175-3182), nude mice, andRag1 and/or Rag2 knockout mice. These mice may optionally be irradiated,or otherwise treated to destroy one or more immune cell type. Thus, invarious embodiments, a genetically modified mouse is provided thatcomprises a humanization of at least a portion of an endogenousnon-human IL-15 locus, and further comprises a modification thatcompromises, inactivates, or destroys the immune system (or one or morecell types of the immunes system) of the non-human animal in whole or inpart. In one embodiment, modification is, e.g., selected from the groupconsisting of a modification that results in a NOD mice, a SCID mice, aNOD/SCID mice, an IL-2Rγ knockout mouse, a NOD/SCID/γc^(null) mouse, anude mice, a Rag1 and/or Rag2 knockout mice, and a combination thereof.In a specific embodiment, the mouse comprises a replacement of allmature IL-15-coding sequence with human mature IL-15 coding sequence.

In one embodiment, the mouse comprises a replacement of all matureIL-15-coding sequence with human mature IL-15 coding sequence, and themouse is a NOD mouse. In one embodiment, the mouse comprises areplacement of all mature IL-15-coding sequence with human mature IL-15coding sequence, and the mouse is a SCID mouse. In one embodiment, themouse comprises a replacement of all mature IL-15-coding sequence withhuman mature IL-15 coding sequence, and the mouse is a NOD/SCID mouse.In one embodiment, the mouse comprises a replacement of all matureIL-15-coding sequence with human mature IL-15 coding sequence, and themouse comprises an IL-2Rγ knockout. In one embodiment, the mousecomprises a replacement of all mature IL-15-coding sequence with humanmature IL-15 coding sequence, and the mouse is a NOD/SCID/γc^(null)mouse. In one embodiment, the mouse comprises a replacement of allmature IL-15-coding sequence with human mature IL-15 coding sequence,and the mouse is a nude mouse. In one embodiment, the mouse comprises areplacement of all mature IL-15-coding sequence with human mature IL-15coding sequence, and the mouse comprises a Rag1 and/or Rag2 knockout.

Genetically modified non-human animals that comprise a modification ofan endogenous non-human IL-15 locus, wherein the modification comprisesa human nucleic acid sequence encoding at least a portion of a matureIL-15 protein, are provided. Although genetically modified cells arealso provided that comprise the modifications described herein (e.g., EScells, somatic cells), in many aspects and embodiments the geneticallymodified non-human animals comprise the modification of the endogenousIL-15 locus in the germline of the animal.

Genetically modified non-human animals that comprise a replacement of anon-human IL-15 gene sequence with a human IL-15 gene sequence areprovided. In various embodiments, an endogenous non-human IL-15 locus ismodified in whole or in part to comprise human nucleic acid sequenceencoding at least one protein-coding exon of a mature IL-15 protein. Invarious embodiments, the human sequence is a human genomic sequence,e.g., a contiguous human genomic sequence comprising one or more exonsthat encode a portion of a mature IL-15 protein, or, e.g., a cDNA thatencodes at least one or more exons that encode a portion of a matureIL-15 protein. In various embodiments, all IL-15 protein-coding exonsthat encode protein sequences that appear in a mature human IL-15protein are humanized. In various embodiments, the humanized IL-15 locusis under control of upstream endogenous regulatory sequences (e.g., allendogenous sequences upstream of the humanization). In variousembodiments, the humanization comprises a human 3′UTR.

In various embodiments, the non-human animals are mammals. In certainembodiments, the mammals are rodents. Rodents that comprise ahumanization of an IL-15 gene, at an endogenous rodent IL-15 locus, areprovided. Methods for making rodents, e.g., mice, that comprise areplacement of an endogenous IL-15 gene or fragment thereof (e.g., afragment comprising one or more exons) with a humanized IL-15 gene, orfragment thereof (e.g., a fragment comprising one or more exons), at theendogenous IL-15 locus. Cells, tissues, and mice are provided thatcomprise the humanized gene are provided, as well as cells, tissues, andmice that express human IL-15 from an endogenous non-human IL-15 locus.Rodents that express a human IL-15 protein under control of anendogenous rodent promoter are also provided.

IL-15 was discovered as an IL-2-independent T cell growth factor thatstimulates T cell proliferation and supports thymic development andnatural killer (NK) cell development (Burton, J. D. t al. (1994) Alymphokine, provisionally designated interleukin T and produced by ahuman adult T-cell leukemia line, stimulates T-cell proliferation andthe induction of lymphokine-activated killer cells, Proc. Natl. Acad.Sci. USA 91:4935-4939; Grabstein, K. H. et al. (1994) Cloning of a TCell Growth Factor That Interacts with the β Chain of the Interleukin-2Receptor, Science 264:965-968). IL-2 and IL-15 share receptor subunits.However, the independent importance of IL-15 in maintenance of immunecell populations is undisputed; IL-15/IL-15R knockout mice exhibit lowCD8+ T cells, low memory CD8+ T cells, and low NK cells, as well asother cell types (reviewed in Steel, J. C. et al. (2012) Interleukin-15biology and its therapeutic implications in cancer, Trends inPharmacological Sciences, 33(1):35-41).

IL-15 is known to be expressed in endothelial cells; IL-15 derived fromendothelial cells stimulates transendothelial migration of T cells (see,Oppenheimer-Marks, N. (1998) Interleukin 15 is Produced by EndothelialCells and Increases the Transendothelial Migration of T Cells in Vitroand in the SCID Mouse-Human Rheumatoid Arthritis Model In Vivo, J. Clin.Invest. 101(6):1261-1272). Thus, early work established a likelihoodthat T cell recruitment to inflammatory sites is mediated by IL-15(Id.). This fact is significant, because improper or over-expression ofIL-15 may readily lead to a pathological phenotype—a situation facedwith transgenic non-human animals that express dysregulated IL-15.Proper IL-15 regulation is important, because IL-15 is believed to be apro-inflammatory cytokine that is at the apex of a pro-inflammatorycytokine cascade, preceding expression of many inflammation mediators(McInnes, I. B. et al. (1997) Interleukin-15 mediates T cell-dependentregulation of tumor necrosis factor-α production in rheumatoidarthritis, Nature Med. 3:189-195, quoted in Waldmann, T. A. (2006) Thebiology of interleukin-2 and interleukin-15: implications for cancertherapy and vaccine design, Nature Rev. Immunol. 6:595-601).

Transgenic mice expressing human IL-15 under control of anenterocyte-specific promoter (T3b promoter) to express human IL-15 inintestinal epithelial cells develop spontaneous inflammation in theduodeno-jejunal area (Yokoyama, S. et al. (2008) Antibody-mediatedblockade of IL-15 reverses the autoimmune intestinal damage intransgenic mice that overexpress IL-15 in enterocytes, Proc. Natl. Acad.Sci. USA 106(37)15849-15854; Ohta, N. et al. (2002) IL-15-dependentactivation-induced cell death-resistant Th1 type CD8 alpha beta+NK1.1+ Tcells for the development of small intestinal inflammation, J. Immunol.169:460-468). See, also, Nishimura, H. et al. (2005) A novelautoregulatory mechanism for transcriptional activation of the IL-15gene by nonsecretable isoform of IL-15 generated by alternativesplicing, FASEB J. 19:19-28 (transgenic mice with a randomly insertedmIL-15 gene variant).

Mice transgenic for a secretable isoform of IL-15 under control of anMHC class I promoter have been prepared, but they overexpress IL-15(Yajima, T. et al. (2001) Memory phenotype CD8(+) T cells in IL-15transgenic mice are involved in early protection against a primaryinfection with Listeria monocytogenes, Eur. J. Immunol. 31(3):757-766).Overexpression of IL-15 is correlated with destruction of intestinalepithelium by IL-15-activated cytotoxic T lymphocytes in celiac disease(Yokoyama, S. et al. (2011) Transgenic Mice that Overexpress Human IL-15in Enterocytes Recapitulate Both B and T Cell-Mediated PathologicManifestations of Celiac Disease, J. Clin. Immunol. 31:1038-1044),presumably due to promoting proliferation of CD8+ T cells that targetenterocytes through NKG2D (natural killer group 2, member D)-mediatedprocess that include cognate receptors such as MICA/B (Id., at 1039). Itseems clear by now that locally-expressed IL-15 causes T cell-mediatedtissue damage in the intestine in celiac disease (Id.).

At least one study of transgenic mice that are engineered to overexpressIL-15 in muscle tissue and in circulation (employing a skeletal musclepromoter) establish that IL-15 overexpression affects metabolism; suchmice appear to employ IL-15 as a myokine that reduces body fat andprovides resistance to diet-induced adiposity (Quinn, L. S. et al.(2009) Oversecretion of interleukin-15 from skeletal muscle reducesadiposity, Am. J. Physiol. Endocrinol. Metab. 296:E191-E202).

IL-15 is also thought to be implicated in rheumatoid arthritis, perhapsthrough abnormal T-cell infiltration of joints (reviewed in, e.g.,Fehninger T. A. and Caligiuri, M. A. (2001) Interleukin 15: biology andrelevance to human disease, Blood 97(1):14-32). Sarcoidosis patientsalso produce alveolar macrophages that express IL-15, which may mediateT-cell proliferation in lung (Id., at 23). IL-15 may also mediate organrejection in allografts via proliferation of T cells (Id., at 24). IL-15may also be implicated in adult T-cell leukemia (e.g., HTLV-1-mediated),based at least in part on the activation of IL-15-mediated pathways inpatients with adult T-cell leukemia (Id.). In vitro work suggests thatIL-15 activates HIV replication, which may be the case in humans as well(Id., at 25).

In transgenic mice that express IL-15 driven by an MHC class I promoterinfected with Mycobacterium bovis bacillus Calmette-Guérin,overproduction of IL-15 rendered the mice susceptible to LPS-inducedlethal liver injury, an effect that was not observed when CD8+ T cellswere depleted from the mice (Yajima, T. (2004) Overexpression ofInterleukin-15 Increases Susceptibility to Lipopolysaccharide-InducedLiver Injury in Mice Primed with Mycobacterium bovis bacillusCalmette-Guérin, Infection and Immunity 72(7):3855-3862), suggesting aneffect mediated by IL-15 overproduction.

Transgenic mice that express IL-15 driven by a skeletal muscle promoterexhibited a higher insulin sensitivity and a resistance to diet-inducedobesity, and appeared to promote fatty acid metabolism (Quinn, L. S. etal. (2011) Overexpression of interleukin-15 in mice promotes resistanceto diet-induced obesity, increased insulin sensitivity, and markers ofoxidative skeletal muscle metabolism, International Journal ofInterferon, Cytokine and Mediator Research, 3:29-42).

Selective blockade of murine IL-15 has been studied, including a solubleIL-15Rα, which may be of clinical benefit in controlling rheumatoidarthritis (Id., at 27). Thus, non-human animals that express human orhumanized IL-15, including in a physiologically relevant fashion, areuseful for assessing or identifying selective blockers of human IL-15.According to one reviewer, “the development of effective human IL-15blocking agents . . . with in vivo blocking activity could facilitaterapid translation of such approaches to the clinic” (Id., at 27). Thus,genetically modified non-human animals, e.g., non-human animals thatcomprise a human IL-15 gene in their germline, wherein the non-humananimals express human IL-15 in a physiologically appropriate manner,would be quite useful.

IL-15 is a pleiotropic cytokine that is required for NK cell developmentand function and T cell homeostasis. It is particularly important forthe memory CD8+ T cell compartment. IL-15 is produced primarily bydendritic cells and macrophages, and is transpresented via IL-15/IL-15Rcomplex to NK cells and T cells. IL-15 is also known to be apro-inflammatory cytokine that induces production of other cytokines,recruits and activates T-cells and other inflammatory cells, promotesdevelopment and survival of NK cells, and promotes angiogenesis; andmany of these features are displayed in psoriatic lesions (reviewed andreported in Villadsen, L. S. (2003) Resolution of psoriasis uponblockade of IL-15 biological activity in a xenograft mouse model, J.Clin. Invest. 112(10):1571-1580). It has been proposed that IL-15 is atthe apex of the pro-inflammatory cytokine cascade, with variousstrategies under way to modulate IL-15 signalling for disease treatment(reviewed in Waldman (2006) The biology of interleukin-2 andinterleukin-15: implications for cancer therapy and vaccine design,Nature Reviews Immmunology, 6:595-601). In a xenograft mouse model ofpsoriasis in SCID mice, blockade of IL-15 using an antibody to IL-15R(or IL-15) resulted in reduction of severity of psoriasis (Id.). Thus,non-human animals that express IL-15 in a physiologically relevantmanner are useful (e.g., have a well-established utility) in models forhuman diseases, including but not limited to models inimmune-compromised mice, such as, e.g., SCID mice and otherimmune-compromised mice. Thus, in one embodiment, a rodent (e.g., amouse) comprising a human or humanized IL-15 gene under control ofendogenous non-human regulatory elements (e.g., a humanization of thecoding gene for IL-15 in a rodent, e.g., a mouse) is provided.

The IL-15 gene is found on human chromosome 4q31 and on mouse chromosome8. The human gene contains 8 exons (7 coding), and appears to exist intwo isoforms in both humans and mice (see, e.g., Fehninger T. A. andCaligiuri, M. A. (2001) Interleukin 15: biology and relevance to humandisease, Blood 97(1):14-32). mRNA for IL-15 is produced in a widevariety of tissues and cell types, and regulation of the IL-15 gene inhumans appears to be negatively regulated by an upstream region whosedeletion results in a dramatic increase in IL-15 promoter activity (Id.,at 17). Transgenic mice that lack posttranscriptional control of IL-15exhibit a fatal lymphocytic leukemia (Id.). Regulation of IL-15expression appears to be very tight, mediated at least by 5′untranslated region AUG triplets, 3′ regulatory elements, and a putativeC-terminal region regulatory site (reviewed in McInnes, I. B. andGracie, J. A. (2004) Interleukin-15: a new cytokine target for thetreatment of inflammatory diseases, Current Opinion in Pharmacology4:392-397). The human IL-15 gene has nine exons and eight introns, whichincludes an exon 4a that is present in humans but not mice, though themature IL-15 protein is encoded by just exons 5 through 8 (reviewed inBudagian, V. et al. (2006) IL-15/IL-15 receptor biology: A guided tourthrough an expanding universe, Cytokine & Growth Factor Reviews17:259-280). There are two alternatively spliced mRNA products thatproduce two IL-15 isoforms, which differ only in the length of signalpeptide, which is true for both mouse and human proteins (see, e.g.,Id.). FIG. 1 , which depicts a humanization strategy fora mouse IL-15locus, omits upstream mouse sequences that were not humanized (includingexons that do not appear in the mature protein) for simplicity, andpresents a re-numbering of exons relevant to the humanization shown.

IL-15 is expressed in many cell types and tissues, including monocytes,macrophages, dendritic cells, keratinocytes, epidermal cells,fibroblasts, and epithelial cells of nerve, kidney, placenta, lung,heart, and muscle (Grabstein, K. H. et al. (1994) Cloning of a T CellGrowth Factor That Interacts with the β Chain of the Interleukin-2receptor, Science 264:965-968).

Mouse IL-15 coding sequences were humanized as depicted in FIG. 1 ,which omits depiction of two non-coding exons (which were not humanized)far upstream from the coding exons. 12299 nucleotides of mouse sequencewas replaced by 12896 nucleotides of human sequence to humanize theIL-15 gene.

In one embodiment, the humanized IL-15 locus lacks a human IL-15 5′UTR.In one embodiment, the humanized IL-15 locus comprises a rodent 5′UTR.In a specific embodiment, the rodent is a mouse, and the humanized IL-15locus comprises a mouse IL-15 5′UTR.

Rodents that express human or humanized IL-15 protein, e.g., in aphysiologically appropriate manner, provide a variety of uses thatinclude, but are not limited to, developing therapeutics for humandiseases and disorders. IL-15 antagonists, such as, e.g., soluble formsor IL-15 receptor, can prevent development of collagen-mediatedarthritis in an animal model (see, Ruchatz, H. et al., (1998) SolubleIL-15 receptor α-chain administration prevents murine collagen-inducedarthritis: a role for IL-15 in development of antigen-inducedimmunopathology, J. Immunol. 160:5654-5660;); anti-IL-15 antibodies haveexhibited efficacy against a variety of diseases, including psoriasisand rheumatoid arthritis; in an animal model of arthritis, an IL-15receptor antagonist prevents both the development and progression ofarthritis, as well as reducing lymphocyte infiltration of joints(Ferrari-Lacraz, S. et al. (2004) Targeting IL-15 Receptor-Bearing Cellswith an Antagonist Mutant IL-15/Fc Protein Prevents Disease Developmentand Progression in Murine Collagen-Induced Arthritis, J. Immunol.173:5815-5826); IL-15-mediated signaling has also been implicated inIBD, SLE, inflammatory synovitis, diabetes mellitus, and asthma(reviewed in Budagian, V. et al. (2006) IL-15/IL-15 receptor biology: Aguided tour through an expanding universe, Cytokine & Growth FactorReviews 17:259-280).

Studies with IL-15 knockout mice establish that IL-15 is necessary forthe development of certain immune cells, in particular, NK cells(reviewed in Lodolce, J. P. (2002) Regulation of lymphoid homeostasis byinterleukin-15, Cytokine & Growth Factor Reviews, 13:429-439). Indeed,IL-15 knockout mice do not long survive exposure to certain pathogens(e.g., Vaccinia virus), presumably due to lack of NK and CD8+ T cells(Id.). Thus, effects of hIL-15 antagonists on human NK cell functionrepresent an important application of a humanized IL-15 animal.

In various aspects, genetically modified animals are provided thatexpress human or humanized IL-15, which are useful for testingantagonists to human IL-15. The genetically modified animals may furthercomprise an animal model of a human disease, e.g., the disease isinduced genetically (a knockin or knockout) or otherwise. In variousembodiments, the genetically modified non-human animals further comprisean impaired immune system, e.g., a non-human animal genetically modifiedto sustain or maintain a human xenograft, e.g., a human solid tumor or ablood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor).

EXAMPLES Example 1: Humanizing the Mouse IL-15 Locus

Mouse ES cells were modified to replace certain mouse IL-15 genesequences with certain human IL-15 gene sequences at the endogenousmouse IL-15 locus, under control of mouse IL-15 regulatory elements,using VELOCIGENE® genetic engineering technology, to produce a humanizedlocus as shown in FIG. 1 . FIG. 1 does not show upstream (with respectto direction of transcription of the IL-15 gene) the 5′ untranslatedexons of the mouse gene; Ex1 of FIG. 1 shows a small untranslated region(unfilled) upstream of coding exon. As shown the humanization at thebottom of FIG. 1 , mouse coding exons 1 and 2 were retained, whereasmouse coding exons 3 through 6 were replaced with human exons 3 through6. At the downstream end, human exon 6 is followed by a stop codon and ahuman 3′-UTR, and further by human sequence found downstream of thehuman 3′UTR. For selection purposes, a selection cassette (floxed forremoval by Cre) was included. The humanized locus of FIG. 1 expresses amature IL-15 protein that is fully human.

Targeting Construct. Bacterial homologous recombination (BHR) isperformed to construct a large targeting vector (LTVEC) containingsequences of the human IL-15 gene for targeting to the mouse IL-15 locususing standard BHR techniques (see, e.g., Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech. 21(6):652-659) andgap repair BHR. Linear fragments are generated by ligating PCR-generatedhomology boxes to cloned cassettes followed by gel isolation of ligationproducts and electroporation into BHR-competent bacteria harboring thetarget bacterial artificial chromosome (BAC). Mouse BAC PRCI23-203P7 isused as the source of mouse sequence; human BAC RP11-103B12 is used asthe source of human IL-15 gene sequence. Following a selection step,correctly recombined clones are identified by PCR across noveljunctions, and by restriction analysis. A large targeting vector (LTVEC)containing homology arms and human IL-15 gene sequences was made. MouseES cells were electroporated with the LTVEC constructs, grown onselection medium, and used as donor ES cells to make humanized IL-15mice comprising a replacement at the endogenous mouse IL-15 locus withhuman sequence as depicted in FIG. 1 .

The mouse IL-15 gene (mouse GeneID: 103014; RefSeq transcript:NM_008357.2; ensemble eID:16168) is modified by using genomiccoordinates for deletion GRCM38: ch 8: 82331173-82343471 (minus strand);genomic coordinates for replacement GRCh37: ch4: 142642924-142655819(plus strand). 12299 nucleotides of mouse sequence was replaced by 12896nucleotides of human sequence. The replacement of mouse IL-15 sequenceas described above is graphically presented in FIG. 1 .

The LTVEC comprising the humanized IL-15 gene had about 13 kb ofupstream mouse targeting arm flanked upstream with a MluI site, and a 27kb downstream mouse targeting arm flanked downstream with an AscI site.The LTVEC was linearized with MluI and AscI for electroporation.

Following construction of the LTVEC, nucleotide sequence of the LTVECacross the mouse/human 5′ junction, and human/mouse 3′ junction is asshown in FIGS. 2-4 .

Following electroporation of the ES cell, a loss of native allele assay(see, e.g., Valenzuela et al. (2003)) is performed to detect loss ofendogenous IL-15 sequence due to the targeting.

Correctly targeted ES cells (MAID 5217) were further electroporated witha transient Cre-expressing vector to remove the Neo drug selectioncassette. The resultant cassette-deleted ES cells were designated MAID5218.

Example 2: Humanized IL-15 Mice

Generating humanized IL-15 mice. Donor mouse ES cells comprising ahumanized IL-15 locus (e.g., MAID 5217 or MAID 5218) are introduced intoearly stage mouse embryos by the VELOCIMOUSE® method (Poueymirou et al.(2007) F0 generation mice fully derived from gene-targeted embryonicstem cells allowing immediate phenotypic analyses, Nat Biotechnol25:91-99). Heterozygous mice are obtained, and to obtain homozygoteswith respect to humanized IL-15, heterozygotes are bred.

Example 3: Phenotyping Humanized IL-15 Mice

Mice. Mice were either wild-type (WT) 8-10 week old Balb/c females orage-matched MAID 5217 (heterozygous for human IL-15 gene) females.Alternatively, mice were either wild-type (WT) or age-matched MAID 5217or MAID 5218 (both heterozygous for the human IL-15 gene) mice.

In vivo poly I:C injection. WT Balb/c or MAID 5217 het were injectedwith 50 μg poly I:C (Invivogen; Cat #tlrl-pic) via tail-vein (IVinjection). After 24 hours, mice were sacrificed and bled via cardiacpuncture and serum isolated. Spleens were also harvested and splenocytesprepared by mechanically disrupting spleens through a 70 μM mesh filterfollowed by ACK lysis buffer (Invitrogen) treatment to lyse red bloodcells (RBCs). Isolated splenocytes were cultured for additionalstimulation (see below). Serum was analyzed for human IL-15 using theR&D Systems human IL-15 QUANTIKINE™ kit. WT or MAID 5218 het mice wereinjected with 50 μg poly I:C (Invivogen; Cat #tlrl-pic) via IP. Micewere bled the next day via cardiac puncture and serum was analyzed forhuman IL-15 by ELISA (R&D Systems QUANTIKINE™ ELISA kit).

Bone marrow-derived dendritic cell (BM-DC) preparation. Bone marrow wasflushed from the tibia of non-injected mice and RBCs lysed with ACKlysis buffer. Cells were washed with RPMI complete (w/HEPES, Gentamicin,sodium pyruvate, L-glutamine, and non-essential amino acids)+10% fetalbovine serum (FBS) and counted. 2×10⁶ cells were cultured per well in a6-well plate with 3 mL/well of RPMI complete +10% FBS+50 ng/mL murineGM-CSF+50 ng/mL murine IL-4. Cells were cultured at 370 C/5% CO2 andgiven fresh GM-CSF/IL-4 at days 2 and 4 of culture. At day 5 of culture,non-adherent BM-DCs were harvested from the cultures and respectiveculture media saved (conditioned media).

Splenocyte culture. Spleens were harvested from respective mice andsplenocytes prepared by mechanically disrupting spleens through a 70 μMmesh filter followed by ACK lysis buffer (Invitrogen) treatment to lyseRBCs. Isolated splenocytes were cultured in a 48-well plate at 2×10⁶/mLsplenocytes in 1 mL of RPMI complete+10% FBS. Cells were treated with 10μg/mL poly I:C, 10 μg/mL PMA or left untreated. Cells were cultured assuch overnight and the next day supernatant harvested and concentrated8-fold using Amicon 2 mL filters with 3 kd molecular weight cut-off(MWCO). Concentrated supernatants were analyzed for human IL-15 usingthe R&D systems human IL-15 QUANTIKINE™ kit.

BM-DC culture. 2×10⁶/mL BM-DCs were plated in a 24-well plate in 0.5 mLof fresh RPMI complete+10% FBS and 0.5 mL of conditioned media. Cellswere treated with 25 μg/mL poly I:C, 1 μg/mL LPS or left untreated. Allconditions were performed in duplicate. Cells were cultured as such for36 hrs and then the supernatant harvested. Supernatants wereconcentrated 7-fold using Amicon 2 mL filters with 3 kd MWCO. HumanIL-15 levels in concentrated supernatants were analyzed using the R&Dsystems human IL-15 QUANTIKINE™ kit. RNA was isolated from cells viaRNAeasy™ mini prep kit from Qiagen for RT-PCR analysis of human IL-15transcript levels.

ELISA. R&D systems human IL-15 QUANTIKINE™ kit was used to measure humanIL-15 in serum and concentrated splenocytes or BM-DC supernatants. Kitwas used according to manufacturer's instructions. Additional controlswere performed to validate this kit for specificity (only detects human,not mouse, IL-15) and to confirm it does not react to poly I:C.Therefore, 1000 μg/mL of murine IL-15 was run on the ELISA (note:highest standard for the human IL-15 is 250 μg/mL) and poly I:C alone at25 μg/mL and 12.5 μg/mL. The kit was found to react specifically tohuman IL-15 (no detection of mouse IL-15) and did not react to poly I:C.

RT-PCR. cDNA was prepared from ˜200 ng of isolated RNA usingSUPERSCRIPT™ III First-strand synthesis system for RT-PCR kit(Invitrogen) according to manufacturer's instructions. Specific humanIL-15 transcript was amplified via using Taqman DNA polymerase with thefollowing primers: hIL-15 Forward primer: gtaaraagtg atttgaaaaaaattgaagat (SEQ ID NO:7); hIL-15 Reverse primer: tacaaaactc tgcaaaaattctttaatat (SEQ ID NO:8). PCR reaction was performed with 40 cycles ofthe following: denaturing at 940 C for 15 seconds, annealing at 60° C.for 30 seconds, extending at 72° C. and reaction then kept at 4° C.Transcript was run on a 1% agarose gel using Promega 6× loading dye.

Results. Human IL-15 was observed in serum of poly I:C-injected MAID5217 het, but not in a poly I:C-injected age/sex-matched WT Balb/c mouse(FIG. 6 ; and FIG. 10 , right panel). Similarly, human IL-15 wasobserved in serum of poly I:C-injected MAID 5218 het, but not in a polyI:C-injected age/sex-matched WT mouse (FIG. 10 , left panel). The levelof IL-15 produced in MAID 5218 was comparable to MAID 5217 (FIG. 10 ).PMA-stimulated splenocytes from MAID 5217 secrete low levels of humanIL-15 in vitro (none observed in splenocytes from WT mice).

Additionally, BM-DCs derived from MAID 5217 het demonstrate human IL-15secretion upon in vitro stimulation with poly I:C (TLR3 agonist) and LPS(TLR4 agonist), as well as significant basal levels. RT-PCR analysisdemonstrated specific human IL-15 transcript only in BM-DCs from MAID5217 het mice.

Overall, the data indicates that MAID 5217 het and MAID 5218 hetexpresses human IL-15.

Example 4: Mouse Homozygous for Human IL-15

Heterozygous mice are bred, then genotyped as described above.Homozygous hIL-15 mice are maintained by inbreeding.

1.-22. (canceled)
 23. A method of maintaining human xenograft in amouse, comprising (i) engrafting human tumor cells into a geneticallymodified mouse, wherein the genome of the mouse comprises a replacementof a mouse genomic fragment of a mouse IL-15 gene at an endogenous mouseIL-15 locus with a human genomic fragment of a human IL-15 gene to forma humanized IL-15 gene, the mouse genomic fragment comprises sequencesof exons 3, 4, 5 and 6 of the mouse IL-15 gene coding for a mature mouseIL-15 polypeptide, the human genomic fragment comprises exons of thehuman IL-15 gene coding for a mature human IL-15 polypeptide, and theexons in the human genomic fragment consist of exons 3, 4, 5 and 6 ofthe human IL-15 gene, the humanized IL-15 gene is under control ofendogenous mouse IL-15 upstream regulatory elements at the endogenousmouse IL-15 locus, and the mouse comprises a human IL-15 polypeptide inserum following treatment of said mouse with Poly I:C; and (ii)maintaining the human tumor cells in the mouse.
 24. The method of claim23, wherein the human tumor cells are solid tumor cells or blood cancercells.
 25. The method of claim 23, wherein the humanized IL-15 geneencodes a protein which comprises the amino acid sequence of SEQ ID NO:5.
 26. A method for identifying an agent that is an antagonist of humanIL-15, comprising (i) administering a candidate agent to a geneticallymodified mouse, wherein the genome of the mouse comprises a replacementof a mouse genomic fragment of a mouse IL-15 gene at an endogenous mouseIL-15 locus with a human genomic fragment of a human IL-15 gene to forma humanized IL-15 gene, the mouse genomic fragment comprises sequencesof exons 3, 4, 5 and 6 of the mouse IL-15 gene coding for a mature mouseIL-15 polypeptide, the human genomic fragment comprises exons of thehuman IL-15 gene coding for a mature human IL-15 polypeptide, and theexons in the human genomic fragment consist of exons 3, 4, 5 and 6 ofthe human IL-15 gene, the humanized IL-15 gene is under control ofendogenous mouse IL-15 upstream regulatory elements at the endogenousmouse IL-15 locus, and the mouse comprises a human IL-15 polypeptide inserum following treatment of said mouse with Poly I:C; and (ii)determining whether the candidate agent has an effect on a human IL-15mediated function, and identifying the candidate agent as an IL-15antagonist if it antagonizes the human IL-15 mediated function.
 27. Themethod of claim 26, wherein the human IL-15 mediated function islymphocyte development, and wherein said determining comprises (i)measuring lymphocyte number of the mouse at one or more time pointsfollowing administration of the candidate agent to the mouse, and (ii)determining whether the candidate agent reduces the lymphocytepopulation.
 28. The method of claim 26, wherein the human IL-15 mediatedfunction is lymphocyte infiltration of a tissue or joint, and whereinsaid determining comprises (i) measuring lymphocyte infiltration of thetissue or the joint at one or more time points following administrationof the candidate agent to the mouse over a period of time, and (ii)determining whether the candidate agent reduces lymphocyte infiltrationof the tissue or the joint.
 29. The method of claim 26, wherein thehuman IL-15 mediated function is arthritic progression, and wherein saiddetermining comprises determining whether the candidate agent affectsprogression of an induced arthritis in the mouse.
 30. The method ofclaim 26, wherein the humanized IL-15 gene encodes a protein whichcomprises the amino acid sequence of SEQ ID NO:
 5. 31. A targetingnucleic acid construct comprising a human genomic fragment comprisesexons of a human IL-15 gene coding for a mature human IL-15 polypeptide,and the exons in the human genomic fragment consist of exons 3, 4, 5 and6 of the human IL-15 gene, wherein the human genomic fragment is flankedby 5′ and 3′ mouse homology arms that mediate homologous recombinationand integration of the human genomic fragment into a mouse IL-15 genelocus, such that a genomic fragment of a mouse IL-15 gene at the mouseIL-15 gene locus is replaced with the human genomic fragment to form ahumanized IL-15 gene, wherein the mouse genomic fragment comprisessequences of exons 3, 4, 5 and 6 of the mouse IL-15 gene coding for amature mouse IL-15 polypeptide, and wherein the humanized IL-15 gene isunder control of endogenous mouse IL-15 upstream regulatory elements atthe endogenous mouse IL-15 locus.